Methylglyoxal Attenuates Mycobacterium avium subspecies paratuberculosis (MAP)-Induced Pro-Inflammatory Macrophage Programming Associated with NRF-2 Antioxidant Responses and Reduced MCT4/Lactate-Linked Inflammatory Markers.

Crohn's disease (CD) is a chronic inflammatory bowel disease with a rising incidence and prevalence worldwide. It is associated with Mycobacterium avium subspecies paratuberculosis (MAP). Current CD treatment strategies are based on anti-inflammatory therapies, including anti-TNF-α drugs. These treatment options provide short-term benefits and are associated with numerous side effects in CD patients. Manuka honey is distinguished from other honey by its high content of methylglyoxal (MGO). MGO, a reactive metabolite, is also generated endogenously in macrophages during infection through glycolysis; however, the amount is insufficient to neutralize the ongoing infection and subsequent tissue damage. This study examined whether exogenous, low-dose MGO can modulate MAP-driven inflammatory and glycolysis- and lactate-associated markers in infected macrophages. THP-1 macrophages were infected with the CD-associated MAP strain and then treated with MGO doses at defined time intervals. We measured markers of M1-/M2-like phenotype polarization, monocarboxylate transporters, lactate export, antioxidant responses, cytokines, and selected glycolysis- and lactate-associated markers at both the mRNA and protein levels. MGO reduced M1 signaling markers CXCL10 (p < 0.05), TNF-α (p < 0.0001), IL-1β (p < 0.01), and IL-6 (p < 0.0001). Simultaneously, MGO promoted M2 shift, elevating CD206 by 1.20-fold and IL-10 by 7-fold. Low-dose MGO administration was associated with increases in Nrf-2 (1.4-fold), HO-1 (1.4-fold), and IL-1Ra (1.5-fold), while the pro-inflammatory cytokines decreased. Metabolically, MGO downregulated MCT4 (p < 0.01) and reduced lactate export by 30%. These changes were coupled with higher PHD2 (1.4-fold) and decreases in GLUT1 (0.9-fold), PKD1 (0.8-fold), and IL-1β, consistent with attenuated glycolysis- and lactate-associated inflammatory signaling. These results suggest that hormetic concentration of MGO mitigates MAP-induced inflammatory activation while altering glycolysis- and lactate-related signaling markers in infected macrophages. Most importantly, we unraveled the predicted molecular mechanism by which MGO suppresses inflammation and modulates oxidative damage.